Two‐Hybrid Fluorescence Cross‐Correlation Spectroscopy Detects Protein–Protein Interactions In Vivo

Abstract

Fluorescence cross-correlation spectroscopy (FCCS) uses the correlated motion of two distinct fluorophores to detect their interaction. Whereas FCCS has been used with chemically or genetically labeled interaction partners in vitro, FCCS has never been demonstrated in vivo between two autofluorescent proteins. At least one reaction partner was always chemically labeled. Fos and Jun, two components of the AP-1 transcription factor, are known to exert their function as a dimer and can therefore serve as a reference for dimer formation. Expressing fusion proteins between Fos and the enhanced green fluorescent protein (EGFP), as well as Jun and the monomeric red fluorescent protein 1 (mRFP1) in HeLa cells, we show here, for the first time, in vivo FCCS detection of protein-protein interactions. The mobility of the dimerized species is slow, indicating that DNA-binding might stabilize dimerization. The technique has rich potential applications for the rapid screening of protein-protein interactions in vivo, which are able to clarify events during the whole life of cells.

Time-resolved luminescence resonance energy transfer imaging of protein–protein interactions in living cells

“…Thus, in addition to enhanced sensitivity, LRET imaging displays increased time resolution that may allow analysis of interaction dynamics that cannot be resolved by traditional steady-state FRET or FRET-FLIM. Whereas other optical methodologies such as induced translocation of fluorescent protein fusions (7-9) and FCCS (4,5) can monitor interactions in cells with much better time resolution (0.1-1 s for FCCS), these methods do not provide a spatial map of interactions.…”

“…However, cell-free studies and screening assays do not provide information about the spatio-temporal organization of protein networks in the natural environment of the living cell or organism. A variety of optical methods are available for monitoring protein interactions in cells, including fluorescence cross correlation spectroscopy (FCCS) (4,5), bimolecular fluorescence complementation (6), translocation-based assays (7)(8)(9), and methods that detect intermolecular Förster resonance energy transfer (FRET). Among these methods, only FRET allows dynamic and reversible imaging of protein-protein interactions while simultaneously preserving information about their subcellular distribution (10)(11)(12).…”

High throughput FRET analysis of protein–protein interactions by slide‐based imaging laser scanning cytometry

“…Shortly, FRETexperiments were carried out using HeLa cells transfected with fluorescent proteins. Construction of the expression vectors pSV-c-Fos-ECFP, pSV-c-Jun-EYFP, and the positive control, pSV-ECFP-EYFP (coding for the fusion of the two fluorescent proteins connected by a RNPPVAT linker) is described elsewhere (26). pSV-Fos 215 -ECFP is a truncated version of full-length Fos-ECFP, where the last 164 amino acids have been deleted (23).…”

“…Because of their propensity to form stable heterodimers via a leucine-zipper, Fos and Jun are often used as positive controls in dimerization studies. Earlier, we demonstrated their stable heterodimer formation by FCCS (26) and FRET (23) in live cells, and described the C-terminal conformation of the complex using a Fos-GFP 1 Jun-mRFP1 pair.…”

Cross-talk-free Fluorescence Cross-Correlation Spectroscopy by the Switching Method

“…This method can detect the coincidence of two molecules labeled with fluorophores of different colors in a small detection area on which two laser beams are focused. FCCS has been applied to studies of molecular interactions in homogeneous solutions (Kettling et al, 1998;Doi et al, 2002;Collini et al, 2005;Swift et al, 2006) and in living cells (Saito et al, 2004;Baudendistel et al, 2005;Kim et al, 2005). Unique features of FCCS technology have recently been reviewed by Bacia et al (2006) with emphasis on its application to living cells.…”